Integrin, a cell surface glycoprotein, is an adhesion molecule that functions mainly as a receptor for cell adhesion to extracellular matrices (collagen, laminin and the like) and members of the immunoglobulin family (ICAM-1, VCAM-1 and the like), and mediates signal transduction from extracellular matrices. Thereby, cells receive signals from the extracellular matrices, and differentiation, proliferation, cell death and the like are induced. Integrin is a hetero-dimer consisting of the two subunits α chain and β chain; there are different α chains and β chains occurring in a wide variety of combinations, and there are 24 members of the integrin superfamily. Integrin-knockout mice are fatal or diseased irrespective of which subunit is lacked, suggesting that individual integrins are necessary for the maintenance of life. Therefore, integrin, which transmits information on ambient conditions to cells to stimulate their responses, are thought to function in all situations of biological phenomena, and to mediate a broad range of pathologic conditions.
As such, integrin is indispensable to the survival of organisms, and is thought to play roles even in diseased states; some cases have been reported in which their inhibition helps improve pathologic conditions. For example, an inhibitor of platelet-specific integrin αIIbβ3 has been approved as a therapeutic drug for PCTA restenosis known as abciximab (trade name: ReoPro; Eli Lilly). Natalizumab (trade name: Antegren; ELAN Company), an α4β1 (VLA4) inhibitor, has been approved as a therapeutic drug for multiple sclerosis. The αvβ3 inhibitor Vitaxin (MEDIMMUNE Company) is under development in clinical studies for its neovascularization inhibitory action, osteoclast activation inhibitory action and the like.
Integrin α9β1 is expressed in macrophages, NKT cells, dendritic cells, and neutrophils, and reportedly plays important roles in the infiltration and adhesion of these inflammatory cells, bone resorption and the like. Recently, it has been reported that integrin α9β1 is involved in osteoclast formation, and its involvement in bone destruction has been suggested (Non-patent Document 1). Known ligands thereof include truncated osteopontin (N-terminal OPN), VCAM-1, Tenascin-C and the like. Clinically, significantly elevated levels of integrin and have been observed in the synovial tissues of patients with rheumatoid arthritis (Non-patent Document 2).
Therefore, a monoclonal antibody that binds specifically to α9 integrin protein to act to inhibit α9 integrin-dependent cell adhesion, if developed, would be useful in the diagnosis, prevention or treatment of various diseases involved by α9 integrin in their pathogenesis.
Antibodies that have been reported to exhibit function inhibitory action on human α9 integrin are the mouse monoclonal antibody Y9A2 (Non-patent Document 3), and 1K11, 24I11, 21C5 and 25B6 (Patent Document 1) and 28S1 (Patent Document 2). In vitro experimental results have shown that these antibodies are capable of suppressing human α9 integrin-dependent cell adhesion. Among those, since Y9A2 inhibits cell adhesion to both osteopontin and Tenascin-C, it is considered most promising as a candidate for an antibody drug against α9 integrin.
It should be noted, however, that Y9A2 is a mouse-derived antibody prepared by immunizing a mouse with an antigen, and therefore, direct administration thereof to human is practically impossible from the aspects of safety (induction of antigenicity) and effectiveness (shortened half-life). Therefore, a modification to convert the antibody to a molecule having an amino acid sequence of human antibody while maintaining the activity of Y9A2, i.e., humanization, needs to be performed.
At present, as a production method of humanized antibody, a method based on the method including grafting of amino acid of complementarity determining region (hereinafter sometimes to be indicated as CDR) as designed by Winter et al. (non-patent document 4) is most general. It is also well known here that simultaneous grafting of not only CDR but also non-CDR amino acid involved in the structural maintenance of CDR or binding with an antigen, i.e., a framework region (hereinafter sometimes to be indicated as FR), from a foreign antibody to be the donor of CDR amino acid to a human antibody to be the acceptor of CDR is important for the reproduction of the inherent activity of the donor antibody (non-patent documents 4 and 5).
However, production of a humanized antibody based on CDR grafting includes several problems. Firstly, the most general problem is that even an appropriate selection of FR amino acid necessary for reproduction of the activity of a donor antibody cannot eliminate the difficulty of obtaining a humanized antibody having affinity to an antigen and biological activity exceeding those of the donor antibody.
In recent years, a large number of chimeric antibody, humanized antibody and human antibody has been placed in the market as monoclonal pharmaceutical products. The effective dose of any of them is extremely high and is several mg per 1 kg body weight. Therefore, antibody pharmaceuticals are inevitably expensive, which in turn increases economical burden on the patients and medical costs. The major factors defining the effective dose of an antibody drug include affinity of the antibody to an antigen and the amount of the antigen present in the body. From such aspects, particularly, an improvement in the affinity of an antibody to an antigen leads to a reduction in the dose, and is an extremely useful improvement also resulting in the reduction of economical burden on the patients and medical costs.
To realize an improved affinity of an antibody to an antigen, a method including introduction of amino acid substitution into a variable region of the antibody is often adopted. However, when antibody and antigen are different, the sequence and steric structure of CDR amino acid, as well as the position of amino acid involved in antigen-antibody interactions also vary. Therefore, it is practically impossible to define the position of FR amino acid to be grafted together with CDR as being applicable to any antibody.
Another problem is that, while the whole CDR amino acid of a donor mouse antibody is generally grafted to a template human antibody in the preparation of a humanized antibody based on CDR grafting, an amino acid sequence of CDR derived from a mouse antibody, which is important for binding with an antigen, sometimes shows antigenicity against human, often causing generation of an anti-idiotype antibody.
That is, for the production of a humanized antibody, selection of an appropriate acceptor antibody and selection of CDR amino acid and FR amino acid to be substituted are indispensable for imparting an activity higher than that of a donor antibody while avoiding generation of antigenicity in human and lowered stability of the antibody. These require considerable ingenuity and trial and error.    patent document 1: WO 2006/075784    patent document 2: WO 2008/007804    non-patent document 1: Journal of Bone and Mineral Research, 2006, 21: 1657-1665    non-patent document 2: The Journal of Clinical Investigation, 2005, 115: 1060-1067    non-patent document 3: Am. J. Respir. Cell Mol. Biol., 1996, 15: 664-672    non-patent document 4: Science, 239, 1534-1536 (1988)    non-patent document 5: Proc. Natl. Acad. Sci. USA, 86, 10029-10033 (1989)